The long-term goal of my lab has been an understanding of the structure and function of clathrin-coated membranes in cellular function. In addition to plasma membrane coated pits which mediate endocytosis, clathrin coats are also present on vacuolar and tubular domains of endosomes where they are involved in sorting to late endosomes, or to retrograde or recycling pathways, respectively, through mechanisms that are only partially understood. As the early/sorting endosome is a nexus for virtually all extracellular materials entering cells as well as many intracellular trafficking cargoes, understanding sorting mechanisms and structures in the endosomal region is a key challenge. During the past award period, we have identified and characterized distinct clathrin coated structures in the endosomal region that exhibit extremely rapid but localized movement, termed Gyrating- or G-clathrin, and have shown that they accumulate endocytic cargo destined to be recycled to the plasma membrane. To gain further information about the structure and function of these novel, ubiquitous structures and their role in receptor recycling three inter-related aims are proposed: 1. Does G-clathrin mediate direct release of cargo to the plasma membrane, and/or undergo interconversion with other clathrin-coated membranes in the cell periphery? Rapid, simultaneous 2-color fluorescence imaging using widefield, TIRF and confocal microscopy, separately and in rapid alternation, and using conventional and photo-activatible or -switchable probes for increased temporal and spatial resolution, will be employed to test these hypotheses. Further, in collaboration with G. Raposo (Institut Curie, Paris) we will continue ongoing correlative light- and electron microscopy work to reveal the location and ultrastructural morphology of G- clathrin structures in COS-1 and MNT-1 cells. 2. GGA proteins are a component of G-clathrin and we have found that through its VHS domain GGA1 binds specifically and directly to the class II PI-3-Kinase-C2 (PI3K- C2). As we have also shown that PI3K-C2 binds clathrin and interacts with dynactin and the dynein motor, we hypothesize that this interaction regulates G-clathrin dynamics and function in the cell. This question will be addressed by identifying the loci on each protein responsible for the GGA-PI3K-C2 binding interaction, constructing mutants lacking these determinants, and testing them for their effect on G-clathrin. 3. Preliminary evidence indicates that clathrin light chains are essential for G-clathrn formation and function, novel as these highly conserved proteins are not known to have clear in vivo functions. We will examine the role of light chains and dissect the involvement of their domains and identified binding partners in G-clathrin formation and function in recycling pathways.